Hemostasis is the mechanism by means of which living beings respond to a haemorrhage and involves the participation of two processes that become functional immediately after a lesion and remain active for a long period of time. The first of them is known as primary hemostasis and is characterized by the occurrence of vasoconstriction at the vascular lesion site and platelet aggregate formation. The second one is known as secondary hemostasis, being the phase in which the fibrin clot is formed due to the action of the different coagulation cascade proteolytic enzymes.
Several cofactors and proteolytic enzymes participate in the second phase of the blood coagulation process, all referred to as coagulation factors, and it consists of several phases ending with fibrin formation from fibrinogen hydrolysis due to the action of thrombin. Thrombin is previously formed by proteolytic hydrolysis of an apoenzyme, prothrombin. This proteolysis is carried out by the activated coagulation Factor X (FXa), which binds to the surface of the activated platelets and only in the presence of its cofactor, activated coagulation Factor V (FVa), and calcium ions, and is able to hydrolyze prothrombin. Coagulation Factor X (FX) activation can occur in two separate pathways, the intrinsic pathway and the extrinsic pathway.
The intrinsic pathway consists of a series of reactions in which each proenzyme is hydrolyzed, yielding its active protease form. In each step, the recently formed proteolytic enzyme will catalyze activation of the following proenzyme to successively yield the active form.
In the blood coagulation extrinsic pathway, the Tissue Factor (TF), exposed on adventitia cells at the lesion site, binds to circulating coagulation Factor VII/activated coagulation Factor VII (FVII/FVIIa) to form the TF::FVIIa complex and, in the presence of calcium, to act as a substrate so that FX activation takes place. The extrinsic pathway is currently considered the most relevant pathway in blood coagulation, and it is accepted that in the event of a hemorrhage produced by a vascular lesion, coagulation is triggered due to extrinsic pathway activation involving the interaction of TF with its ligand, FVII/FVIIa.
It has been broadly accepted that TF is the main element responsible for the quickness with which coagulation is initiated, and it is required for FX activation, which in turn begins prothrombin hydrolysis.
Purification of TF has been reported from various tissues such as: human brain, bovine brain; human placenta; ovine brain; and lung. It is widely accepted that while there are differences in structure of TF protein between species there are no functional differences as measured by in vitro coagulation assays.
It is widely accepted that in order to show biological activity, TF must be associated with phospholipids in vitro. It has been shown that the removal of the phospholipid component of TF, for example by use of a phospholipase, results in a loss of its biological activity in vitro.
WO2008080989 describes tissue factor-bearing yeast derived microvesicles comprising a yeast membrane and a tissue factor protein and the use thereof as pro-coagulant agents in the treatment of hemorrhages in a subject.
WO2006004675 describes the expression of tissue factor in plant cells, crude extracts obtained from plants expressing TF and artificial vesicles comprising recombinant TF obtained from plant cells.
EP19359021 describes the expression of tissue factor in insect cells as well as relipidated TF which contains recombinant TF expressed in insect cells.
However, there is a need in the art for additional pro-coagulant preparations based on TF.